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OPEN
received: 07 June 2016 accepted: 28 September 2016 Published: 18 October 2016
Dnmt1 regulates the myogenic lineage specification of muscle stem cells Renjing Liu1,2,3, Kun-Yong Kim1, Yong-Wook Jung1,4 & In-Hyun Park1 DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity. Epigenetic modification of the chromatin plays crucial roles in maintaining cellular states, genomic stability, ensuring proper gene transcription and DNA repair1,2. Methylation of cytosine residues in DNA is carried out by a class of enzymes, the DNA methyltransferases (Dnmts), that tightly regulate the initiation and the maintenance of these methyl marks3,4. Dnmt3a and Dnmt3b are responsible for establishing the de novo patterns of methylation during embryogenesis, while Dnmt1 is responsible for the propagation of methylation patterns. Errors in this enzymatic machinery have profound effects on development and disease5. All three Dnmts are required for embryonic development, with previous studies reporting Dnmt1−/− and Dnmt3b−/− mice to be embryonically lethal6,7. While Dnmt3a−/− mice survive to full term, they die around 4 weeks of age7. Surprisingly, embryonic stem cells (ESCs) can be derived without Dnmts and maintain their stem cell characteristics8. Studies have also highlighted critical roles for DNA methylation in adult stem cells. Loss of Dnmt1 in neuronal progenitors leads to global genomic hypomethylation and neonatal death9, while Dnmt1−/− fibroblasts undergo growth arrest and widespread apoptosis10. In the hematopoietic system, Dnmt3a and Dnmt3b deficiency leads to defects in self-renewal of hematopoietic stem cells, but had no effect on cellular differentiation11. However, the loss of Dnmt1 resulted in self-renewal and differentiation abnormalities in the same cell type12. Studies in the mammalian epidermis reported a premature differentiation of epidermal progenitor cells and tissue loss in the absence of Dnmt1, further emphasizing a role of DNA methylation in maintaining the undifferentiated progenitor cell state13. In skeletal muscle, the importance of DNA methylation in the control of myogenesis was shown in the regulation of master myogenic transcriptional factor, MyoD. MyoD is selectively expressed in skeletal muscle cells and its expression in non-muscle cells is suppressed by DNA methylation14. Demethylating agents such as 5-azacytidine can induce MyoD transcription and lead to myogenic conversion of non-muscle cells such as in the CH310T1/2 and NIH3T3 cell lines15–17. These studies suggest that epigenetic marking through DNA methylation plays a significant role in determining cell fate during muscle development. Limited data exists on the function of Dnmts during myogenesis. It has been reported that DNMT1 mRNA expression in human myoblasts decreased with differentiation, and this coincided with increases in myogenic differentiation gene expressions13. While these results are consistent with the notion that DNA methylation in self-renewal of skeletal muscle stem cells exists, more detailed analyses are needed to formally establish this. 1
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT, 06520. Agnes Ginges Laboratory for Diseases of the Aorta, Centenary Institute, University of Sydney, Camperdown, 2042, Australia. 3Sydney Medical School, University of Sydney, Sydney, 2006, Australia. 4Department of Obstetrics and Gynecology, CHA Gangnam Medical Center, CHA University, Seoul, Republic of Korea. Correspondence and requests for materials should be addressed to I.-H.P. (email: [email protected])
2
Scientific Reports | 6:35355 | DOI: 10.1038/srep35355
1
www.nature.com/scientificreports/
Figure 1. Dnmt1 knockdown adversely affects myotube formation. (A) Dnmt1 expression following differentiation of C2C12 cells. *P
OPEN
received: 07 June 2016 accepted: 28 September 2016 Published: 18 October 2016
Dnmt1 regulates the myogenic lineage specification of muscle stem cells Renjing Liu1,2,3, Kun-Yong Kim1, Yong-Wook Jung1,4 & In-Hyun Park1 DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity. Epigenetic modification of the chromatin plays crucial roles in maintaining cellular states, genomic stability, ensuring proper gene transcription and DNA repair1,2. Methylation of cytosine residues in DNA is carried out by a class of enzymes, the DNA methyltransferases (Dnmts), that tightly regulate the initiation and the maintenance of these methyl marks3,4. Dnmt3a and Dnmt3b are responsible for establishing the de novo patterns of methylation during embryogenesis, while Dnmt1 is responsible for the propagation of methylation patterns. Errors in this enzymatic machinery have profound effects on development and disease5. All three Dnmts are required for embryonic development, with previous studies reporting Dnmt1−/− and Dnmt3b−/− mice to be embryonically lethal6,7. While Dnmt3a−/− mice survive to full term, they die around 4 weeks of age7. Surprisingly, embryonic stem cells (ESCs) can be derived without Dnmts and maintain their stem cell characteristics8. Studies have also highlighted critical roles for DNA methylation in adult stem cells. Loss of Dnmt1 in neuronal progenitors leads to global genomic hypomethylation and neonatal death9, while Dnmt1−/− fibroblasts undergo growth arrest and widespread apoptosis10. In the hematopoietic system, Dnmt3a and Dnmt3b deficiency leads to defects in self-renewal of hematopoietic stem cells, but had no effect on cellular differentiation11. However, the loss of Dnmt1 resulted in self-renewal and differentiation abnormalities in the same cell type12. Studies in the mammalian epidermis reported a premature differentiation of epidermal progenitor cells and tissue loss in the absence of Dnmt1, further emphasizing a role of DNA methylation in maintaining the undifferentiated progenitor cell state13. In skeletal muscle, the importance of DNA methylation in the control of myogenesis was shown in the regulation of master myogenic transcriptional factor, MyoD. MyoD is selectively expressed in skeletal muscle cells and its expression in non-muscle cells is suppressed by DNA methylation14. Demethylating agents such as 5-azacytidine can induce MyoD transcription and lead to myogenic conversion of non-muscle cells such as in the CH310T1/2 and NIH3T3 cell lines15–17. These studies suggest that epigenetic marking through DNA methylation plays a significant role in determining cell fate during muscle development. Limited data exists on the function of Dnmts during myogenesis. It has been reported that DNMT1 mRNA expression in human myoblasts decreased with differentiation, and this coincided with increases in myogenic differentiation gene expressions13. While these results are consistent with the notion that DNA methylation in self-renewal of skeletal muscle stem cells exists, more detailed analyses are needed to formally establish this. 1
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT, 06520. Agnes Ginges Laboratory for Diseases of the Aorta, Centenary Institute, University of Sydney, Camperdown, 2042, Australia. 3Sydney Medical School, University of Sydney, Sydney, 2006, Australia. 4Department of Obstetrics and Gynecology, CHA Gangnam Medical Center, CHA University, Seoul, Republic of Korea. Correspondence and requests for materials should be addressed to I.-H.P. (email: [email protected])
2
Scientific Reports | 6:35355 | DOI: 10.1038/srep35355
1
www.nature.com/scientificreports/
Figure 1. Dnmt1 knockdown adversely affects myotube formation. (A) Dnmt1 expression following differentiation of C2C12 cells. *P
